Doxorubicin-induced Apoptosis Is Associated with Increased Transcription of Endothelial Nitric-oxide Synthase

Abstract

The clinical efficacy of the antitumor antibiotic drug doxorubicin (DOX) is severely limited by its dose-limiting cardiotoxicity in cancer patients. DOX-induced generation of reactive oxygen species was proposed to be a major mechanism of its cardiotoxicity. Previously, we showed that DOX undergoes a reductive activation at the reductase domain of endothelial nitric-oxide syn-thase (eNOS) forming the semiquinone and superoxide (Vá squez-Vivar, J., Martasek, P., Hogg, N., Masters, B. S. S., Pritchard, K. A., Jr., and Kalyanaraman, B. (1997) Biochemistry 36, 11293-11297). In this report, we provide evidence for DOX-induced increase in eNOS transcription and protein expression in bovine aortic endothelial cells (BAEC). We propose that DOX-induced hydrogen peroxide formation is responsible for the increased transcription of eNOS. BAEC treated with anti-sense eNOS oligonucleotide inhibits DOX-induced endo-thelial apoptosis. Treatment with antioxidants restored the levels of antiapoptotic proteins (Hsp70 and Bcl-2) in DOX-treated BAEC. DOX-induced intracellular oxida-tive stress, as measured by oxidation of dichlorodihy-drofluorescein diacetate to dichlorofluorescein and hy-droethidium to ethidium, was inhibited by antisense eNOS oligonucleotide and antioxidant treatment. Furthermore , antiapoptotic antioxidants (e.g. FeTBAP, ebselen, and-phenyl-tert-butyl nitrone) inhibited DOX-induced eNOS transcription. We conclude that DOX-induced apoptosis is linked to the redox activation of DOX by eNOS.